Intestinal epithelial stem cells (IESC) renew the small intestine and colon epithelium throughout life, and are critical to epithelial repair and regeneration after injury. Aging-associated alterations in IESC and ability to renew or repair the intestinal epithelium may predispose to infection, impair digestive and absorptive capabilities, impact susceptibility to colorectal cancer and limit ability to tolerate chemo- or radiation therap. Understanding of age-induced changes in IESC has been hampered by lack of valid IESC biomarkers. This proposal will use a Sox9-EGFP reporter mouse, where distinct levels of Sox9-EGFP expression permit direct visualization and isolation of IESC, progenitors, enteroendocrine cells (EEC) and other differentiated intestinal epithelial cells. Using Sox9-EGFP mice we have direct evidence for aging-associated IESC expansion and that insulin-like growth factor 1 (IGF1) potently and specifically promotes IESC regeneration after injury in young adults. Levels of IGF1, IGF2 or insulin profoundly impact lifespan, and may dictate the survival and numbers of pluripotent or tissue-restricted stem cells for tissue repair and integrity. IGFs and insulin signa through the IGF1 receptor (IGF1R) or the insulin receptor (IR). IR exists as an IR-B isoform, which mediates metabolic effects of insulin and an IR-A isoform that may be particularly relevant to growth. Preliminary data demonstrate expression patterns of IGF1R, IR-A and IR-B which predict distinct roles of these receptors in IESC, progenitors, EEC and other differentiated intestinal epithelial lineages. Mice with complete Villin-Cre (VC)- mediated deletion IR (VC-IR/) or IGF1R (VC-IGF1R/) in intestinal epithelium, cross-bred with Sox9-EGFP mice, will be used test a central hypothesis that IR protects IESC and intestinal epithelium from aging- induced dysfunction by limiting mitogenic anti-apoptotic actions of IGF1R and promoting maintained differentiated function. Aim #1 will define the impact of aging on number, function and transcriptome of IESC, progenitors, EEC or differentiated lineages, and the ability of these aged cells to regenerate after injury Aim #2 will define the impact of IR deletion on IESC and progenitors, EEC, differentiated lineages and response to insulin or IGFs throughout normal aging, or during IESC and epithelial regeneration after injury Aim#3 will define the impact of IGF1R deletion on IESC and progenitors, EEC, differentiated lineages and response to insulin or IGFs throughout normal aging, or during IESC and epithelial regeneration after injury. Findings will fundamentally advance our knowledge of the effects of aging on IESC and identify new mechanisms, biomarkers, and potential therapeutic targets to better monitor and promote successful IESC and intestinal aging. PUBLIC HEALTH RELEVANCE: Insulin and insulin-like growth factors are essential to normal metabolism, growth and tissue repair. Excess levels of these hormones are linked to accelerated aging and aging-associated diseases including diabetes and cancer. This project will identify the receptors that drive beneficial effects of these hormones on renewal and repair of the epithelial lining of the intestine over the course of normal aging and limit their effects o aging-associated dysfunction or diseases of the intestine.